Biomod/2013/Komaba/Experiment

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(Concept)
(Results)
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[[Image:result_final.jpg]]
[[Image:result_final.jpg]]
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<p>We succeeded in making the Cylinder and the Cylinder-Ring and synthesizing DNA Spiders!!!<br>
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<p>'''We succeeded in making the Cylinder and the Cylinder-Ring and synthesizing DNA Spiders!!!'''<br>
As for electrophoresis and other structures, we are still working on for better results!
As for electrophoresis and other structures, we are still working on for better results!

Revision as of 02:15, 27 October 2013


Contents

Concept

 To make our DNA Screw, we started creating a Cylinder and a Ring made of DNA Origami. First, we made a cylinder and a ring separately. Every structure was observed by Atomic Force Microscope; AFM. Although the image of the Ring was unclear, we succeeded in making the Cylinder. Second, we made modified structure of the Cylinder and the Ring. To avoid the electrostatic repulsion between the Cylinder and The Ring, the structure was made from a single scaffold strand, and the Cylinder and the Ring were connected. We observed the structure by AFM. Finally, we synthesized the spiders and attached them to the cylinder; The observation of the structures where DNA Origamis are combined with the DNA spiders is a challenge for the future.

Reagents

TAE bufferTris-acetate-EDTA buffer, which is essential to make DNA Origami.
Mg2+Has a function to shape DNA Origami.
TE bufferHas a function to preserve DNA.
TBE bufferTris-Borate-EDTA buffer, which is essential when making DNA tile and doing electrophoresis.
M13mp18Scaffold strand which we bought from TAKARA BIO INC.

Results

Image:result_final.jpg

We succeeded in making the Cylinder and the Cylinder-Ring and synthesizing DNA Spiders!!!
As for electrophoresis and other structures, we are still working on for better results!

Methods

Cylinder(1st ver.)

We made a Cyliner by DNA Origami.

  • First, we made staple mix for the Cylinder. We put 2.5μL for each staple whose stock is 50μM into the tube and diluted to 250nM with TE buffer. We had a set of 179 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.
  • Second, we made 100μL solution of the Cylinder. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 250nM), and 72.5μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 67.5μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
  • Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.2°C/min.

  • Here, we put the table of amount, final concentration and stock concentration of each protocols.

    Staple Mix

    AmountStock
    Staple2.5μL(each)50μM
    TE buffer52.5μL
    Total500μL


    10-equivalent

    ConcentrationAmount Stock
    Mg2+ 12.5mM 1.25μL 1M
    TAE10x 10μL
    M13 2.5nM 6.25μL 40nM
    Staple Mix25nM 10μL 250nM
    mQ 72.5μL
    Total 100μL



    15-equivalent

    Concentration Amount Stock
    Mg2+ 12.5mM 1.25μL 1M
    TAE10x 10μL
    M13 2.5nM 6.25μL 40nM
    Staple Mix 37.5nM 15μL 250nM
    mQ 67.5μL
    Total 100μL


    Ring (1st ver.)

    We made a Ring by DNA Origami.

    • First, we made staple mix for the Ring. We put 5μL for each staple whose stock is 50μM into the tube and diluted to 500nM with TE buffer. We had a set of 80 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.
    • Second, we made 100μL solution of the Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 500nM), and 66.25μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 61.25μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
    • Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.2°C/min.

    • Here, we put the table of amount, final concentration and stock concentration of each protocols.


      Staple Mix

      Amount Stock
      Staple 5μL(each) 50μM
      TE buffer 100μL
      Total 500μL


      10-equivalent

      Concentration Amount Stock
      Mg2+ 12.5mM 1.25μL 1M
      TAE10x 10μL
      M13 5nM 12.5μL 40nM
      Staple Mix 50nM 10μL 500nM
      mQ 66.25μL
      Total 100μL

      15-equivalent

      Concentration Amount Stock
      Mg2+ 12.5mM 1.25μL 1M
      TAE10x 10μL
      M13 5nM 12.5μL 40nM
      Staple Mix 75nM 15μL 500nM
      mQ 61.25μL
      Total 100μL


      Agarose Gel Electrophoresis(Cylinder(1st ver.) and Ring(1st ver.))

      We also tried Gel Electrophoresis to see whether we had only one structure.

      • First, we prepared 30 mL of gel. We put 0.525 % of Agalose, 3 mL of TBE 10x, 375 μL of Mg2+ (stock concentration: 1M) into the tube and diluted with mQ. After adding all the protocols into the tube, we vortexed for a few seconds.
      • Second, we poured the gel into the frame.
      • Third, we put the frame into the refrigerator and left it for 2 hours.
      • Next, we mixed 10 μL of the following protocols and 2 μL of loading buffer for each protocol. The protocols were the solution of Cylinder (10-equivalient), Cylinder (15-equivalent), Ring (10-equivalent), Ring(10-equivalent), Staple mix of Cylinder and the Ring, and M13.
      • Next, we poured 400 μL of TBE buffer into a container and were subjected to electrophoresis at 50 V for one and a half hour at 4°C.
      • Next, we prepared 10 mL solution of SyBR Green 1 Dye. We put 1 μL of SyBR 1 10000x and 99.9 mL of TBE buffer.
      • We moved the gel to the sharre and poured the solution of SyBR.
      • We waited for 1 minute.
      • We put the sharre on the tharmomixer for 10 seconds.
      • We observed under UV light.
      • Here, we put the amount, final concetration, and stock concentration of the protocols we used for the electrophoresis.

        Concentration Amount
        Agarose 1.75%(wt/V) 0.525g
        TBE10x 1x 3mL
        Mg2+ 12.5mM 375μL
        mQ up to 30mL
        Total 30mL




        Observing Cylinder (1st ver.) by AFM

        Cylinder 15-equivalent
        Cylinder 15-equivalent

        We succeeded in observing the Cylinder!

        The scan rate was 0.996 Hz.


        We used thermo cycler from BioRad MiniOpticon.

        Cylinder-Ring

        We made Cylinder-Ring structure with DNA Origami.

      • First, we made staple mix for the Cylinder-Ring. We put 2μL for each staple whose stock is 50μM into the tube and diluted to 200nM with TE buffer. We had a set of 198 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.
      • Second, we made 50μL solution of the Cylinder-Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 12.5μL of Staple Mix (stock concentration: 200nM), and 25.625μL of mQ into the tube. As for 15-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 18.75μL of Staple mix (stock concentration: 200nM), and 19.375μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
      • Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.1°C/min.

      • Here, we put the table of the amount, final concentration and stock concentration of protocols used in Cylinder-Ring structure.

        Staple Mix

        Concentration Amount Stock
        Staple 200nM 2μL 50μM
        TE buffer 104μL
        Total 500μL

        10-equivalent

        Concentration Amount Stock
        Mg2+ 12.5mM 0.625μL 1M
        TAE10x 5μL
        M13 5nM 6.26μL 40nM
        Staple Mix 50nM 12.5μL 200nM
        mQ 25.625μL
        Total 50μL

        15-equivalent

        Concentration Amount Stock
        Mg2+ 12.5mM 0.625μL 1M
        TAE10x 5μL
        M13 5nM 6.25μL 40nM
        Staple Mix 75nM 18.75μL 200nM
        mQ 19.375μL
        Total 50μL

        Observing Cylinder-Ring by AFM

        cylinder-ring structure
        cylinder-ring structure

        We successfully observed the cylinder-ring structure. A few cylinders and some ring shaped stractures were seen.

        The scan rate was 0.996 Hz.



        Ring (2nd ver.)

        We also made the Ring with DNA tile, just in case.

        • First, we diluted the oligo to 100μM.
        • Second, we made staple mix for the Ring (2nd ver.). We put 10μL for each staple whose stock is 100μM into the tube and diluted to 5μM with TE buffer. We had a set of 12 staples and we had total amount of 200μL. After putting all the protocols into the tube, we vortexed for a few seconds.
        • Third, we made 50μL solution of the Ring (2nd ver.). We put 0.7μL of Mg2+(stock concentration: 1M), 5μL of TBE 10x, 25μL of Staple Mix (stock concentration: 5μM), and 19.3μL of mQ into the tube. After putting all the protocols into the tube, we vortexed for a few seconds.
        • Fourth, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.02°C/min.

        • Here, we put the table of the amount, final concentration and stock concentration of protocols used in Ring (2nd ver.) structure.

          Staple Mix

          Concentration Amount Stock
          Staple 5μM 10μL 100μM
          TE buffer 80μL
          Total 200μL


          Ring (2nd ver.)

          Concentration Amount Stock
          Mg2+ 14mM 0.7μL 1M
          TBE 10x 5μL
          Staple Mix 2.5μM 25μL 5μM
          mQ 19.3μL
          Total 50μL



          Cylinder(first ver.) + Common Footings and DNA Spiders with Four Walking Legs

          This part of our experiment aimed at seeing if the walking legs actually work. Here we used DNA spiders with four walking legs, which are different from the DNA spiders described in the Design. We planned to mix common footing,DNA spider, and the cylinder(1st ver.) after the cylinder was synthesized followed by being annealed and the DNA spider was synthesized. Zn2+ starts the DNA spider's cleaving motion. The interval of the footings on cylinder(1st ver.) is too wide for the DNA spider to cross so once all the four legs cleave the occupied footings that they attach, they will be detached from the cylinder. Therefore, taking "before" picture by AFM in which the DNA spiders are attached on the cylinders' surface and "after" picture in which the spiders disappear on the surface are considered to be a proof of the walking legs' function.

          Synthesis of DNA spider

          The spider was synthesized in the following protocol; 10μL Streptavidin, 11.36μL walking leg, 6.5μL TE, and 6μL NaCl

          Spider

          Amount Stock
          Streptavidin 10μL 1.89µM
          walking legs 11.36μL 100μM
          NaCl 6μL
          TE buffer 6.5μL

          Mixing of Cylinder(first ver.) + common footings and DNA spiders

          4.0 μl of the solution of the cylinder(1st ver.) which had been annealed for three hours and 16.0 μl of the DNA spiders were mixed into one tube and left it for 30 minutes at room temperature(20~25℃). The solution of the cylinder(1st ver.) which had been annealed for 20 minutes also received the same operation.
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